In the blood, iron in its ferric oxidation state is bound to the protein transferrin. Normally, transferrin is only partially saturated with iron and serves to transport iron from the digestive tract to the blood forming organs, where iron is removed from transferrin and incorporated into hemoglobin. Unsaturated iron binding capacity (UIBC) is the measurement of the amount of transferrin that carries no iron, while serum iron is the measurement of the amount of iron that is bound to transferrin.
The assays of serum iron and UIBC are useful in diagnosing, differentiating and predicting various forms of anemia. The measurement of serum iron in combination with the measurement of UIBC allows the physician to determine which part of the body's pathway to hemoglobin synthesis is deficient, and with such knowledge the treatment of the anemia becomes apparent.
Abnormal serum iron and UIBC levels can be used to predict that anemia will occur in certain pregnancies due to poor absorption of folic acid from the digestive tract. The reason that these serum iron/UIBC changes occur before anemia has developed is due to the long half-life of erythrocytes in the blood. The hemoglobin level represents the adequacy of hemoglobin formation over several weeks prior to hemoglobin measurement, and serum iron/UIBC measurements indicate the current status of hemoglobin formation. Therefore, serum iron/UIBC measurements allow therapy to be implemented before anemia develops, thereby preventing injury to the mother or fetus.
Traditionally, serum iron has been assayed by adding a serum sample to a reagent buffered at an acid pH, which dissociated ferric ion from transferrin. The reagent included a reducing agent, which aided in the dissociation process and reduced ferric ion to ferrous ion. A chromogenic reagent was then added and the chromogen complexed with ferrous iron to form a colored complex, which was measured spectrophotometrically.
However, traditional serum iron assays have been problematic for several reasons. First, serum contains chromatic components, such as hemoglobin and bilirubin, which significantly absorb light at the measuring absorption wavelength of the iron-chromogen colored complex. Second, turbidity due to lipemia interferes with spectral measurements of serum iron. Third, reducing agents, such as thioglycollic acid and ascorbic acid, used in the assay are unstable. Fourth, cupric ion found in serum can compete with ferrous ion for complexing with the chromogen. Fifth, some serum components, such as bilirubin, citric acid, oxalic acid, phospholipids and proteins, can bind serum iron, thus preventing serum iron from complexing with the chromogen.
Certain improvements were made to the serum iron assay by Garcic, Clin. Chim. Acta, 94, 115-19 (1979) (hereinafter Garcic). Garcic discloses the use of the ammonium salt of the chromogen chromazurol B (also known as chrome azurol B; eriochrome azurol B; and mordant blue 1) (hereinafter CAB), and the surfactant cetyltrimethylammonium bromide (CTMA), which react with ferric ion to form a colored complex. The CAB-CTMA-iron ternary complex is spectrophotometrically measured at 630 nanometers (nm), a wavelength at which the chromatic components of serum either no longer interferingly absorb light or minimally absorb light (in the case of turbid samples). Further, Garcic claims cupric ion interference is negligible, which is disputed by Torelli (U.S. Pat. No. 4,810,656, col. 1, lines 11-26), and reducing agents are avoided. However, the serum iron assay requires a 20 minute reaction time, which is too long for automation. Although some automated analyzers can perform an assay that requires a reaction time longer than about 10 minutes, the overwhelming majority of commercially available automated analyzers (such as the Hitachi 704, 707, and 747 analyzers, and the Olympus AU 5000) cannot perform an assay that requires a reaction time longer than about 10 minutes, and a reaction time of less than 10 minutes is greatly preferred (see Takano et al., U.S. Pat. No. 4,588,695, col. 3, lines 29-31). Automation of the serum iron assay is highly desirable because it is one of the twenty most commonly performed medical diagnostic tests in clinical laboratories.
Another problem with the Garcic method is that protein interference is substantial. Protein interference must be subtracted from the measurement of a test sample by employing a serum blank comprised of serum, chromogenic reagent and a masking reagent that includes citric acid, which prevents formation of the CAB-CTMA-iron complex.
The Garcic method requires the preparation of the ammonium salt of CAB. Since the acid form of CAB is poorly soluble in water, the ammonium salt of CAB is prepared to impart sufficient solubility to CAB in aqueous solutions. To avoid the necessity of preparing the ammonium salt, Tabacco et al., U.S. Pat. No. 4,407,962 ('962), improved the Garcic method by adding ethanol to the chromogenic reagent to solubilize CAB in its acid form. Therefore, preparation of the ammonium salt of CAB is no longer necessary. However, a 20-minute reaction time is still required for the assay. Further, a serum blank with maskino reaoent is required to compensate for protein interference.
Tabacco et al., U.S. Pat. No. 4,703,015 ('015), discloses use of the chromogen chromazurol S (also known as chrome azurol S; mordant blue 29; and chromeazurol S) (hereinafter CAS) as a substitute for CAB in the chromogenic reagent for a serum iron assay. Although CAS has a slightly lower molar coefficient of extinction (molar absorptivity) than CAB, CAS is more soluble in aqueous medium and the addition of an organic solvent to the chromogenic reagent is not required. However, assay reaction time is 20 minutes; therefore, the assay is not automatable. Further, protein interference remains a problem, thus requiring a serum blank that includes a masking reagent, and interference from cupric ion may still be significant. Torelli. col. 1, lines 11-26.
Torelli disoloses a chromogenic reagent for serum iron assay that eliminates protein and cupric interferences. The reagent includes CAB, a surface active agent (such as CTMA) in a concentration of at least 500 milligrams (mg)/liter (l), a salt (preferably sodium chloride) in a concentration that imparts an ionic strength of at least 100 grams (g)/l (expressed in terms of sodium chloride concentration), and an amino acid, such as glycine, to eliminate cupric ion interference. However, the high salt concentration of this reagent is disadvantageous for automation because many automated analyzers wash and reuse reaction vessels. If sodium chloride is used, any salt residue remaining after washing of the reaction vessel will artificially elevate a subsequent serum analysis for sodium and chloride. Further, the high concentration of surface active agent lowers the molar absorptivity of the CAB-CTMA-iron ternary complex.
Denney et al., U.S. Pat. No. 4,224,034 ('034), and Outcalt et al., U.S. Pat. No. 4,154,929 ('929) disclose the use of dimethylsulfoxide (DMSO) in a serum iron assay to speed up the assay and to diminish turbidity due to lipemia and/or protein precipitation. '034 col. 4, lines 58-61 and col. 8, lines 33-42; '929 col. 4, lines 58-61 and col. 8, lines 38-47. The '034 and '929 patents do not teach the addition of DMSO to eliminate nonturbidity related protein interferences because a serum blank measurement is required by the assay methods. '034 col. 8, lines 43-61; '929 col. 8, lines 48-66. Further, the serum iron assay methods taught by the '034 and '929 patents employ the unstable reducing agent ascorbic acid. Also, the percentage of DMSO employed in the serum iron assay taught by '034 and '929 is 7% (vol.:vol.) in the final reaction mixture (10% vol.:vol. in the acetate buffer used in the assay). '034 col. 8, lines 45-50; '929 col. 8, lines 50-55.